The synthesis of double stranded DNA, complementary to messenger RNA, and the insertion of this DNA into prokaryotic vectors is of fundamental importance to the molecular cloning and analysis of eukaryotic genes. The construction of complementary DNA libraries, i.e., bacteria or bacteriophage containing complementary DNA clones representative of a messenger RNA population, is often essential for a full understanding of gene expression and processing. Accordingly, the construction of directional libraries, in which complementary DNA is synthesized and ligated to a suitable vector, e.g.,, a plasmid or bacteriophage lambda vector, in a predetermined orientation to permit the expression of the inserted DNA, is highly desirable. Further, it is desirable that methods of complementary DNA synthesis permit the insertion of synthesized DNA into bacteriophage lambda vectors as such vectors generally have higher efficiencies than plasmid vectors for DNA library construction. See, Maniatis, T. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, N.Y. (1982).
A profusion of methods are presently available for the construction of complementary DNA libraries. See, e.g., Maniatis, T. et al, supra. However, the process remains difficult to master because methods of DNA library construction usually entail multiple sequential enzymatic reactions on small amounts of substrate. Methods of first strand complementary DNA synthesis relevant to the present invention rely upon the enzymatic synthesis of DNA from a nucleic acid template, e.g., messenger RNA. Enzymes capable of catalyzing the synthesis of DNA are referred to as "RNA dependent DNA polymerases" where the nucleic acid template is RNA and "DNA dependent DNA polymerases" where the template is DNA (generally, however, RNA dependent DNA polymerases are also capable of functioning as DNA dependent polymerases). More specifically, RNA dependent DNA polymerases such as AMV or MMLV reverse transcriptases are relied upon for the enzymatic synthesis of the first strand of complementary DNA from a messenger RNA template. Both types of DNA polymerases require, in addition to a template, a polynucleotide primer and deoxyribonucleotide triphosphates. The synthesis of first strand complementary DNA is usually primed with an oligo-d(T), consisting of 12-18 nucleotides in length, that initiates synthesis by annealing to the poly-A tract at the 3' terminus of eukaryotic messenger RNA molecules. However, other primers, including short random oligonucleotide primers, can be used to prime complementary DNA synthesis. During the polymerase reaction the primer is extended, stepwise, by the incorporation of deoxyribonucleotide triphosphates at the 3' end of the primer. Additionally, for optimal activity, DNA polymerases usually require magnesium and other ions to be present in reaction buffers in well defined concentrations. Following synthesis of the first strand of complementary DNA, several methods can be employed to replace the RNA-template with the second strand of DNA. One such method involves removal of the messenger RNA with NaOH and self-priming by the first strand of complementary DNA for second strand synthesis. Generally, the 3' end of single stranded complementary DNA is permitted to form a hairpin-like structure that primes synthesis of the second strand of complementary DNA by E. coli DNA polymerase I or reverse transcriptase. However, the method most commonly relied upon involves the replacement synthesis of second strand complementary DNA. See, Gubler, U. and Hoffman, B. J. (1983) Gene 25: 263-269. During replacement synthesis the product of the first strand synthesis, a complementary DNA: messenger RNA hybrid, provides a template and a primer for a nick-translation reaction in which the enzyme RNase H produces nicks and gaps in the messenger RNA strand, resulting in a series of RNA primers for synthesis of the second strand of complementary DNA with the enzyme E. coli DNA polymerase I. After synthesis of a double stranded complementary DNA, the synthesized DNA must be introduced into a host cell, e.g., a bacterial strain, and replicated by means of a suitable vector such as a bacteriophage lambda vector. Several methods have been described and are presently available for ligation of double stranded complementary DNA to bacteriophage lambda vectors. See, e.g., Young, R. A., Davis, R. W. (1983) Proc. Natl. Acad. Sci. 80:1194-1198; and Huynh, T. V., Young, R. A., Davis, R. W. (1985) in DNA Cloning Volume III, a practical approach, ed., D. M. Glover, IRL Press. Such methods involve, for example, the addition of short synthetic polynucleotide linkers which are ligated to the ends of the complementary DNA after second strand synthesis. The linkers contain sequences that are recognized by an appropriate restriction endonuclease, e.g., EcoR I. After the linkers have been ligated to the ends of the complementary DNA they are then cut with the appropriate restriction endonuclease to generate identical cohesive DNA sequences on both ends of the complementary DNA, thereby facilitating ligation of the complementary DNA to a bacteriophage lambda vector. Additionally, as complementary DNA clones of interest may contain restriction sites recognized by the restriction endonuclease, complementary DNA must usually be treated with a methylase, such as EcoR I methylase, prior to ligation of the synthetic linkers to protect the internal restriction sites from subsequent digestion. While such methods yield complementary DNA in bacteriophage lambda vectors and protect the complementary DNA of interest, the directionality of the DNA is not preserved. Thus, such methods fail to ensure that the synthesized complementary DNA will be inserted into a vector in the proper orientation to permit expression in a host cell. Alternatively, methods have been described involving the sequential addition of linkers for the construction of complementary DNA libraries that preserve the directionality of the DNA clones of interest. See, e.g., Helfman, D. M., Feramisco, J. R., Fiddes, J. C., Thomas, G. P. and Hughes, S. H. (1983) Proc. Natl. Acad. Sci. USA 80:31-35. Generally, however, presently available methods which preserve the directionality of complementary DNA rely upon methodology which is difficult to perform and tend to be either inefficient or ineffective for the construction of complementary DNA libraries in bacteriophage lambda vectors. As the introduction of a vector into a host strains is one of the least efficient steps in the construction of complementary DNA libraries, the inability to use bacteriophage lambda vectors, which generally have higher efficiencies than plasmid vectors, is an important limitation inherent in such methods.
Recently, a method has been developed for the construction of directional libraries in bacteriophage lambda vectors which involves the priming of first strand complementary DNA synthesis with a linker/primer. See, Krawinkel, U. and Zoebelein, R. (1986) Nucl. Acids Res. 14:1913; and Han, J. H. and Rutter, W. J. (1987) Biochemistry 26:1617-1625. The linker/primer is typically an oligonucleotide containing a restriction site sequence at the 5' end and an oligo-d(T) sequence at the 3' end of the molecule. After synthesis of the first strand of complementary DNA, a primer, containing a restriction site different from the linker/primer, annealed to the end of the complementary DNA. After ligation of the adaptor or second linker, the first restriction site contained within the linker/primer is cleaved, resulting in removal of the adaptor or second linker from the 3' end of the complementary DNA. The product of this method is, therefore, a complementary DNA molecule having different cohesive ends on each end of the molecule. Such a DNA molecule can thereafter be ligated to a vector that has been cleaved to generate compatible cohesive termini, permitting the insertion of the complementary DNA into the vector in an orientation to permit expression. However, a significant limitation inherent in the foregoing method is that it is not possible to protect the complementary DNA of interest from restriction endonuclease digestion without also preventing cleavage of the linker/primer. Unlike methods in which linkers are added after second strand synthesis, the complementary DNA synthesized by the foregoing method cannot be methylated to protect it from digestion prior to addition of the linker/primer as the linker/primer is utilized to initiate first strand synthesis. Further, the treatment of the complementary DNA synthesized by this method would result in the protection of restriction sites within the linker/primer, and subsequent inability to cleave the adaptor or second linker from the 3' end to generate different cohesive termini for purposes of ligation. Therefore, if the double stranded complementary DNA contains one or more restriction sites identical to the restriction site in the linker/primer, the complementary DNA will be cleaved into fragments at the time it is prepared for ligation to a vector and, subsequently, only a portion of the desired DNA will be cloned. This undesired, but unavoidable, cleavage of complementary DNA molecules prior to ligation substantially increases the difficulty of isolating and analyzing complementary DNA molecules. To alleviate the problems discussed above, the selection of a restriction enzyme, and corresponding restriction site in the linker/primer sequence, which rarely cleaves eukaryotic DNA has been suggested. However, the absence of a particular restriction site within the complementary DNA of interest cannot be presumed. Moreover, even rare restriction enzymes, such as Not I and Sal I, will potentially digest the complementary DNA of interest.
Although the problems enumerated above are not intended to be exhaustive, the limitations inherent in the methods presently available for the synthesis of complementary DNA and construction of directional DNA libraries are readily apparent. Accordingly, there exists a need for an improved method of construction of complementary DNA libraries which permits the insertion of complementary DNA in cloning vectors, particularly bacteriophage lambda vectors, in the orientation required for expression and protects the complementary DNA of interest from digestion by restriction endonucleases. Additionally, there exists a need for an improved method for the construction of directional DNA libraries that does not rely upon the presence or absence of specific nucleotide sequences in the complementary DNA of interest. The present invention meets these needs.